the human connexin gene family of gap junction proteins: distinct chromosomal locations but similar...

GENOMICS 10,250-256 (1991)

The Human Connexin Gene Family of Gap Junction Proteins: Distinct Chromosomal Locations but Similar Structures

GLENN I. FISHMAN,*,’ ROGER L. EDDv,t THOMAS B. %ows,t LAWRENCE ROSENTHAL, * AND LESLIE A. LEINWAND$

*Department of Medicine-Division of Cardiology and *Department of Microbiology and Immunology, Albert Einstein College of Medicine, 7300 Morris Park Avenue, Bronx, New York 10461; and tDivision of Human Genetics, Roswell Park

Memorial Institute, New York State Department of Health, 666 Elm Street, Buffalo, New York 74263

Received October 29, 1990; revised January 16, 1991

Connexins are protein subunits that constitute gap junction channels. Two members of this gene family, con- nexin43 (Cx43) and connexin32 (Cx32), are abundantly expressed in the heart and liver, respectively. Human genomic DNA analysis revealed the presence of two loci for Cx43: an expressed gene and a processed pseudogene. The expressed gene (GJAI) was mapped to human chromosome 6 and the pseudogene (GJAZP) to chromosome 5. To determine whether Cx32 was linked to Cx43, somatic cell hybrids were analyzed by polymerase chain reaction and hybridization, resulting in the assignment of the gene for Cx32 (GJBZ) totheXchromosomeatXpll+q22. Compari- son of the structures of connexin genes suggests that members of this multigene family arose from a single precursor, but evolved to distinct chromosomal locations. 0 1991 Academic Press, Inc.

INTRODUCTION

Connexins are membrane-spanning proteins that assemble to form the intercellular channels of gap junctions. By facilitating the transfer of ions and small molecules from cell to cell, these channels are thought to modulate a number of processes, including embryogenesis, differentiation, and electrotonic cou- pling (for reviews see Hertzberg and Johnson, 1988; Beyer et al., 1990). The expression of the various connexin isoforms is tissue-specific and developmentally regulated (Fishman et al., 1991; Beyer, 1990; Dermiet- zel et al., 1989; Gimlich et al., 1990). In addition, the biophysical properties of each connexin, such as the unitary conductance value and gating characteristics, are distinctly different (Fishman et aZ., 1990; Eghbali et al., 1990; Burt and Spray, 1988). Nonetheless, the physiological significance associated with the tem-

1 To whom correspondence should be addressed.

poral and spatial expression of particular connexin isoforms remains unknown.

We have begun characterizing connexin43,2 the isoform expressed in the mammalian heart. Because gap junction channels are critical components of the cardiac conduction system, alterations in connexin43 gene expression may profoundly influence the electro- physiology of the heart. We recently described the molecular cloning, characterization, and functional expression of the human connexin43 cDNA (Fishman et al., 1990). Our initial analysis revealed two highly homologous connexin43 loci. Here we demonstrate that these loci represent the expressed gene and a processed pseudogene. The two sequences are 97% identical in the coding region, and despite numerous base substitutions and a short deletion, the pseudogene maintains a near full-length open reading frame. Us- ing rodent-human somatic cell lines, we have assigned these loci to chromosomes 6 and 5, respectively. We have also mapped the human connexin32 gene to chromosome Xpll-tXq22. Comparison of the structures of several connexin genes suggests that members of this multigene family arose from a common precursor (Miller et al., 1988, Zhang and Nichol- son, 1990).

MATERIALS AND METHODS

Isolation of Genomic Clones

The isolation and characterization of clones encoding the human connexin43 cDNA and a portion of the first intron of the gene have been described previously (Fishman et al., 1990). A library that contained

’ The following symbols have been approved for the genes described in this report: GJAI-connexin43, gap junction protein, (~1 (43 kDa); GJAIP-connexin43 pseudogene, gap junction protein, alpha pseudogene 1; GJBl-connexin32, gap junction protein, pl (32 kDa).

08SS-7543/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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HUMAN CONNEXIN GENE FAMILY 251

Hue111 and AZuI partially digested fragments of human genomic DNA cloned into the EcoRI site of Charon4A was screened with a cDNA probe encom- passing most of the coding region, previously designated HCGJ7. The EcoRI insert of HCGJ7 was radio- labeled utilizing random hexanucleotides and the Klenow fragment of DNA polymerase (Oligolabelling Kit, Pharmacia Fine Chemicals, Piscataway, NJ.) Ap- proximately 1 X 10’ plaques were lifted onto Gene- Screen filters (New England Nuclear, Boston, MA). Filters were hybridized at 42°C in 50% formamide, 5X SSC, 1X Denhardt’s solution, 1% SDS, 100 pg/ml denatured salmon sperm DNA, and 5 X 10’ cpm/ml probe. Filters were washed in 0.2~ SSC with 0.2% SDS for at least 1 h at 65°C. Two positive clones were identified and carried through sequential plating until plaque pure.

Restriction endonuclease mapping of these two clones showed them to be identical and suggested that they did not correspond to the expressed connexin43 gene. Therefore, a second genomic library that contained complete EcoRI digest fragments cloned into the EcoRI site of Charon was screened. Two oligonucleotides corresponding to nucleotides lo-30 (antisense) and nucleotides 112-135 (sense) of the human connexin43 cDNA were end-labeled with [T-~‘PJATP and polynucleotide kinase (Pharmacia Fine Chemi- cals) according to the manufacturer’s directions. Ap- proximately 2 X lo6 plaques were lifted onto Nytran (Schleicher & Schuell, Keane, NH) filters and hybridized in 5X SSC, 50 mM sodium phosphate, pH 7.4,1X Denhardt’s solution, 2% SDS, and 100 pg/ml denatured salmon sperm DNA, along with 1 X 10’ cpm/ml probe. Numerous positive plaques were identified. The filters were then erased and rehybridized with an oligonucleotide corresponding to nucleotides 100-120 (antisense) of the human connexin43 cDNA. A single doubly positive plaque was identified and carried through sequential plating until plaque pure.

DNA Sequence Analysis

Phage DNA from positive clones was purified by the plate lysate method (Maniatis et al., 1982.) and the EcoRI inserts were subcloned into the plasmid vector pTZ19R (Pharmacia Fine Chemicals). Inserts were sequenced by dideoxy chain termination reac- tions, using custom-designed oligonucleotides synthe- sized at the Albert Einstein College of Medicine Shared DNA Synthesis Facility. Sequence data were analyzed using Staden computer software (Pearson and Lippman, 1988).

Southern Blots

Genomic DNA from human, mouse, and mouse X human somatic cell hybrid lines was digested to

completion with EcoRI, electrophoresed on 0.8% TAE-agarose gels, and capillary transferred to nylon membranes (Nytran, Schleicher & Schuell). For detection of connexin43 sequences, filters were hybridized using the HCGJ7 cDNA probe. For detection of connexin32 sequences, a 304-bp cDNA probe was generated using genomic DNA and the polymerase chain reaction, as described below. Hybridization conditions for both cDNA probes were as described above.

Polymerase Chain Reaction

To generate a connexin32-specific cDNA probe, human genomic DNA (100 ng) was mixed with oligonucleotide primers (1 FM final) corresponding to nucleotides 635-686 (sense) and 919-939 (antisense) of the human connexin32 cDNA (Kumar and Gilula, 1986). The reaction mixture included 50 n&f KCl, 10 n&f Tris-HCl, pH 8.5, 1.5 mM MgCl,, 0.1% gelatin, 200 pi?4 (each) dNTPs, and 2.5 U of Taq polymerase (Cetus Corp., Emeryville, CA) in a total volume of 100 ~1. The samples were denatured at 94’C for an initial 7-min period and then cycled by denaturing at 94°C for 30 s, annealing at 60°C for 30 s, and extending at 72°C for 30 s. A final lo-min extension period was added. The single 304-bp reaction product was gel- purified by electrophoresis through a 1.5% agarose gel and then further purified by excising the band and using GeneClean beads (BiolOl, La Jolla, CA) according to the manufacturer’s directions. Approxi- mately 50 ng of the cDNA reaction product was radio- labeled as described above.

For chromosome mapping experiments, PCR analysis of genomic DNA (100 ng) from human, mouse, and various somatic hybrid cell lines was carried out under identical conditions, except that the reaction volume was decreased to 20 ~1. The entire reaction was electrophoresed on a 1.5% agarose gel containing 1 pM ethidium bromide and visualized under ultravio- let illumination. Each lane was scored for the presence or absence of the appropriate 304-bp band.

RESULTS

Two Connexin43 Loci: Gene and Processed Pseudogene

Our previous studies had suggested the presence of two highly homologous human connexin43 genomic sequences (Fishman et aZ., 1990). Southern blot analysis of genomic DNA digested with multiple restriction endonucleases probed with numerous portions of the human connexin43 cDNA consistently revealed two major bands. One cDNA clone that was isolated from a human fetal cardiac library contained an intron, rep- resenting an incompletely processed transcript. A

252

0 5 kb

FISHMAN ET AL.

Exl IVS Ex2

n 1 I I I I 1 EH S P s E

I E

cx-10 HCGJ16

FIG. 1. Partial structure of the human connexin43 gene. Clone Cx-10 (heavy line) contains a 6-kb EcoRI insert which includes 4.8 kb of 5’ flanking sequence, the 0.2-kb first exon, and 1 kb of intervening sequence. Clone HCGJ16 (heavy line), which has been described previously (Ref. (8)), represents an incompletely processed transcript and contains the acceptor splice junction between the first intron and the second exon. The size of the intervening sequence has not been precisely determined. Restriction enzymes E, EcoRI; H, H&II; P, PstI; S, StyI; Exons are shown by rectangles; introns are shown by solid lines. The coding region is cross-hatched. Restriction mapping suggests that exon 2 includes the remainder of the connexin43 gene; however, clone HCGJ16 extends only to the EcoRI site.

probe derived from this intron recognized a single genomic fragment, suggesting that it recognized only the true human connexin43 gene, whereas probes derived from the cDNA recognized an intronless pseudogene as well. Direct genomic cloning and sequence analysis confirm this hypothesis. Two unique clones, designated Cx-6 and Cx-10, were obtained by screening two genomic libraries. Restriction digest analysis of these two X phage clones indicated that they corre- sponded to the two loci identified by Southern blotting.

Cx-10 contained a single 6-kb EcoRI insert, as shown in Fig. 1. This size is consistent with one of the two bands found by hybridizing a probe derived from the 5’ end of the cDNA with genomic DNA (see Fish- man et al., 1990; Fig. 3). Additional restriction digests and sequence analysis demonstrated that this fragment was derived from the expressed connexin43 gene. The fragment contained -4.8 kb of 5’ flanking sequence, the 186-bp first exon, and approximately 1 kb of the first intron. As shown in Fig. 2, the genomic sequence and cDNA are identical until nucleotide 186, at which point a splice junction donor site is evident in the Cx-10 genomic clone. These splice junctions are precisely those predicted by the incompletely processed transcript previously described (Fishman et aZ., 1990) and conform to the GT-AG rule (Breathnach et aZ., 1978).

Cx-6 contained an - 12-kb insert, with restriction endonuclease fragments corresponding in size to those found previously by Southern blotting (Fish- man et al., 1990). Partial sequence analysis, shown in Fig. 2, demonstrated that this clone was highly homologous to the connexin43 cDNA, but lacked the intervening sequence found in the other genomic clone, Cx-10. Numerous base substitutions were found, as well as a single 3-nucleotide deletion in the coding region which maintained the same reading frame.

Overall homology within the coding region was -97%. A poly(A)-rich region was found and aligned precisely with the poly(A) tail found in the cDNA. Based on the discrepancies with the expressed gene, the lack of intervening sequence, and the presence of a poly(A) tract, the Cx-10 locus appears to represent a processed pseudogene. Sequence comparison and alignment of the rat connexin43 cDNA (Beyer et aZ., 1987) with both the Cx-6 and Cx-10 genomic clones demonstrate that the 5’ ends of all three diverge at about the same point. Primer extention studies and anchored PCR analysis (Frohman et aZ., 1988) also support this point of divergence as the major transcription start site in human cardiocytes (data not shown). Based on the assignment of transcription initiation shown in Fig. 2, the Cx-10 genomic clone pro- vides an additional 45 nt of 5’ sequence that are part of the human connexin43 mRNA transcript, but that were not included in the cDNA sequence (8). A TATA-box-like element is seen beginning at nucleotide -30. Surprisingly, the same element is found in the pseudogene, beginning with nucleotide -18.

Connexin43 Loci Reside on Different Chromosomes

The two connexin43 loci were assigned to chromosomes by Southern blot analysis of DNA from mouse X human hybrid cell lines. As shown in Fig. 3, two bands of 3.3 and 6.6 kb are recognized by hybridizing EcoRI-digested human genomic DNA with the HCGJ7 cDNA probe. These bands segregate independently with respect to the human chromosome content of the hybrid cell lines. The lower molecular weight band corresponds to the true connexin43 gene and maps to chromosome 6, whereas the higher molecular weight band corresponds to the connexin43 pseudogene and maps to chromosome 5. The basis for these assignments is summarized in Table 1 (Naylor

HUMAN CONNEXIN GENE FAMILY 253

Lb 9.4 -

4.4 -

2.3- 2.0-

FIG. 3. Southern blot hybridization of somatic cell hybrid genomic DNA. Genomic DNA was prepared from human (H), mouse (M), or 37 cell hybrids derived from 14 unrelated human cell lines and 4 mouse cell lines. Sixteen hybrid lines are shown here (l-16). The membrane was hybridized with the connexin43 coding region probe HCGJ7 (see Materials and Methods). Two EcoRI fragments of 3.3 and 6.6 kb, which segregate independently with respect to the human chromosome content of the hybrid cell lines, are found. The lower molecular weight band corresponds to the true con- nexin43 gene and the higher molecular weight bands corresponds to the connexin43 pseudogene. These loci map to chromosomes 6 and 5, respectively.

et al., 1983; Shows et al., 1978, 1982, 1984; Shows, 1983).

Connexir232 Locus Resides on the X Chromosome

To determine whether functional members of the human connexin gene family reside on the same or different chromosomes, the location of the con- nexin32 gene was also determined. Mouse X human somatic cell hybrid DNA was analyzed by both Southern blotting and the polymerase chain reaction. Through selection of primers specific for the human connexin32 sequence, conditions that generated only the correct 304-bp product when the appropriate human genomic template was encountered were found. As shown in Fig. 4, cell lines harboring the con- nexin32 gene are easily distinguished from those without it. This analysis demonstrated that the con-

FIG. 2. Nucleotide sequence from connexin43-like genes. Par- tial sequence analyses of the human connexin43 gene (H-Cx43 Gene), human connexin43 cDNA (H-Cx43 cDNA), human con- nexin43 pseudogene (H-Cx43 Pseudo), and rat connexin43 cDNA (R-Cx43 cDNA (2)) are shown. Numbering begins with the puta- tive transcription initiation site (A) labeled EXONl and includes only those nucleotides that constitute the mature mRNA. Nucleo- tide identity among sequences is indicated by a colon. Dashes have been inserted for optimal alignment. Sequence from exons is shown in upper case; flanking and intervening sequences are shown in lower case. Presumptive TATA-box-like elements are underlined. Splice junctions conform to the GT-AG rule and are shown in boldface. The 3’ extent of the human cDNA, including the poly(A) addition site and poly(A) tail, is aligned with the corresponding region of the pseudogene. Amino acids encoded by the pseudogene that differ from the human connexin43 protein are shown below the nucleotide sequence in italics.

254 FISHMAN ET AL.

TABLE 1

Segregation of the Connexin43 Gene with Human Chromosomes in EcoRI-Digested Human-Mouse Cell Hybrid DNA on Southern Blots

Human chromosomes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

6.6-kb band

No. of concordant hybrids

+/+ 3 9 8 7 13 5 6 9 4 10 7 9 5 11 6 4 10 11 5 7 11 5 7 -/- 21 18 16 17 24 22 18 11 18 13 16 13 17 11 20 20 7 16 21 11 6 17 9

No. of discordant hybrids

+/- 9 4 3 6 0 8 6 4 9 3 6 4 8 2 7 9 2 2 8 6 2 8 4 -/+ 2 5 7 7 0 2 4 13 6 10 8 11 7 13 4 4 17 8 3 13 18 4 10

% Discordancy 31 25 29 35 0 27 29 46 41 36 38 41 41 41 30 35 53 27 30 51 54 35 47

3.3-kb band


+/+ 15 5 4 5 7 4 6 2 7 5 4 3 7 4 2 6 6 2 5 6 15 -/- 26 21 18 20 22 30 21 14 22 16 20 14 21 13 24 24 9 17 24 15 7 19 12


+I- 4 113 2 0 3 15 0 2 3 4 0 3 5 0 15 2 16 1 -/+ 4 9 10 10 8 0 6 16 8 13 10 16 9 17 6 6 21 13 6 15 23 8 12

% Discordancy 23 28 32 35 27 0 26 46 35 36 32 51 35 46 24 30 58 38 30 46 65 41 43

Note. This table is compiled from data on 37 cell hybrids derived from 14 unrelated human cell lines and 4 mouse cell lines. The hybrids were characterized by karyotypic analysis and by mapped enzyme markers. The DNA probe was hybridized to Southern blots containing EcoRI-digested DNA from the human-mouse hybrida. The scoring was determined by the presence (+) or absence (-) of human bands in the hybrids on the blots. The scoring was compared to the presence or absence of human chromosomes in each hybrid. A 0% discordancy indicates a matched segregation of the probe bands with a chromosome. The two main bands of the probe for connexin43 on EcoRI blots segregated independently. By somatic cell hybrids, the 6.6-kb band mapped to human chromosome 5 and the 3.3-kb band mapped to human chromosome 6.

nexin32 mapped to the X chromosome. This locus was confirmed by standard Southern blot analysis, specifically to Xpll-tq22, as summarized in Table 2.

DISCUSSION

In this study, we have mapped the chromosomal loci of all members of the human connexin gene family identified to date. This includes the connexin43 and connexin32 genes, as well as the connexin43 processed pseudogene described herein. In addition, we have characterized genomic clones containing the 5’ end of the connexin43 gene and a complete con- nexin43 processed pseudogene. In other species, such as Xenopus, chick, and mouse, cDNA clones that en- code additional isoforma have been isolated (reviewed in Beyer et al., 1990), suggesting that further members of the human connexin gene family may yet be identified. The high stringency conditions used during our library screening and genomic hybridiza-

tions make it unlikely that other connexin43-like genes exist; however, reduced stringency screenings would potentially identify less homologous isoforms.

L I 2 3 4 5 6 7 8 9 IO II 12 I3 14 15 16M H L

FIG. 4. Polymerase chain reaction analysis of somatic cell hybrid DNA. Genomic DNA was prepared as described above and 50 ng from each cell line was amplified with human connexin32-specific oligonucleotide primers (see Materials and Methods). A 304- bp product is evident in the human cell line (H) and in those hybrids (lanes 5.7-11, 15-16) that contain the human connexin32 locus.

255 HUMAN CONNEXIN GENE FAMILY

TABLE 2

Segregation of Connexin32

Human chromosomes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

In human-mouse cell hybrid DNA with human chromosomes by PCR


+/+ 2 5 7 8 7 5 9 6 3 10 9 10 5 11 5 4 10 5 3 9 9 1 14 -/- 7 4 3 5 5 6 4 4 7 2 4 6 4 2 4 6 13 7 4 3 7 8


+/- 12 11 8 8 9 11 6 10 13 6 7 6 11 5 10 12 5 11 13 7 7 15 0 -/+ 14 6 4 4 3 5 5 17 5 3 5 7 5 3 8 6 2 5 6 2 0

% Discordancy 59 62 58 48 52 56 46 60 58 52 48 36 64 48 62 60 54 68 60 48 52 68 0

With human chromosomes in EcoRI-digested human-mouse cell hybrid DNA on Southern blots


+/+ 2 4 9 7 6 5 10 7 1 10 9 9 7 10 4 5 10 5 3 9 10 3 14 -/- 13 10 8 12 8 12 9 10 10 5 9 9 10 4 9 11 4 7 11 6 7 9 10


-+I- 14 12 7 9 10 11 5 9 15 6 7 7 9 6 10 11 6 11 13 7 6 13 0 -/+ 15 6 3 7 3 6 5 3 9 6 6 5 11 5 4 11 8 4 9 8 5 0

% Discordancy 50 55 43 39 55 45 37 45 62 50 42 42 45 55 54 48 55 61 55 52 45 60 0

Note. The data for connexin32 by PCR are from 25 cell hybrids and the those for connexin32 by Southern blot analysis are from EcoRI digests of 31 cell hybrids, as described above. The probe for connexin32 on EcoRI blots and on PCR analysis segregated with the X chromosome. By PCR data from the hybrid DUA-1A with the X/15 translocation Xqter+Xpll::15qll-15qter, connexin32 localized to Xpll-tXqter. By Southern blots with the translocation hybrids ATR-13 (-) 5pter+5q35::Xq22+Xqter, DUA-IA (+) Xqter+Xpll:: 15qll+15 qter, DUA-ICSAZB (-) 15pterhl5qll: :Xpll-*Xpter, REX-11BSHF (-) 22pter+22q13::Xq22*Xqter, XOL- 6 (-) lpter+lql2::XqZB+Xqter, and XTR 3BSAgB (-) 3pter-*3q21::Xq28+Xqter, the connexin32 gene mapped to Xpll-rXq22.

The physiological need for tissue-specific and developmentally regulated expression of members of the connexin gene family is not well understood. Further- more, pathophysiologic states that result from abnormalities in connexin gene expression have not been identified. Conceivably, many defects in connexin gene expression are lethal, thus complicating the rec- ognition and isolation of genetic mutants. The use of restriction fragment length polymorphisms and or- ganisms more amenable to genetic analysis, such as Drosophila, may enable us to identify phenotypes that result from abnormalities of connexin gene expression. We have already screened a panel of 13 normal humans and found no evidence of connexin43 gene or pseudogene polymorphisms (data not shown). How- ever, the mapping data presented in this report may be useful in linkage studies of phenotypes that potentially arise from abnormalities within or near the connexin loci. We are currently attempting in situ chromosome hybridizations to map these loci more finely.

The three human connexin genes studied in this report all map to different chromosomes. Thus, members of this multigene family are not linked in a

fashion similar to the sarcomeric myosin heavy chain gene family (Leinwand et al., 1983). Interestingly, the structures of the human connexin43 and rat con- nexin32 genes (Miller et al., 1988) are remarkably similar. In both genes, the first exon is quite small (186 and 35-90 bp, respectively) and contains only 5’ un- translated sequence. A large intervening sequence is followed by a second exon that contains the entire coding region. Furthermore, the initiation codon begins with nucleotide 17 of the second exon in both genes. A similar intron-exon structure is reportedly found in the mouse connexin26 gene as well (Zhang and Nicholson, 1990). These features suggest that all connexin genes arose by duplication of a common precursor.

The structure and sequence of the connexin43 processed pseudogene suggest that it arose by a retropo- son-mediated event. The 5’ and 3’ ends of the pseudogene align closely with the mRNA transcript. Further- more, the pseudogene lacks intervening sequence and displays a poly(A) addition site homolog followed by a poly(A)-rich region. Finally, the site of integration of the pseudogene is unrelated to the true connexin43

256 FISHMAN ET AL.

gene. The presence of the same TATA-box-like eie- ment both in the gene and flanking the pseudogene is of note. Although unlikely, we have not ruled out the possibility of transcription from the pseudogene. While a number of base substitutions and a short deletion are found in the pseudogene coding region, an essentially full-length protein is encoded; thus it will be of interest to express and analyze functionally the channel behavior of the encoded protein.

Using a luciferase reporter gene system, we have observed that the first 500 bp of 5’ flanking sequence directs transcription in HUH-7 cells, a human hepa- toma line that expresses abundant connexin43. The isolation of 5’ flanking sequence for the human con- nexin43 gene will enable us to define those regulatory elements that confer tissue-specific and developmentally regulated expression upon this gene.

ACKNOWLEDGMENTS

G. I. Fishman is the recipient of a Physician-Scientist Award (lKllHL02391) from the NIH and a Grant-in-Aid from the Ameri- can Heart Association, New York City Affiliate. This work was also supported in part by NIH Grants HL37412 to L. A. Leinwand and HG00333 to T. B. Shows.

Note added inproof. Since submission of this paper, similar chromosomal localizations have recently been reported by Willecke et al. (1990).

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REFERENCES

BEYER, E. C. (1990). Molecular cloning and developmental expression of two chick embryo gap junction proteins. J. Biol. Ch.em. 266: 14439-14443.

BEYER, E. C., PAUL, D., AND GOODENOUGH, D. A. (1987). Connexin43: A protein from rat heart homologous to a gap junction protein from liver. J. Cell Biol. 105: 2621-2629.

BEYER, E. C., PAUL, D. L., AND GOODENOUGH, D. A. (1990). Connexin family of gap junction proteins. J. Memb. Biol. 116: 187-194.

BREATHNACH, R., BENOIST, C., O’HARE, K., GANNON, F., AND CHAMBON, P. (1978). Ovalbumin gene: Evidence for a leader sequence in mRNA and DNA sequences at the exon- intron border. Proc. Natl. Acad. Sci. USA 75: 4853-4857.

BURT, J. M., AND SPRAY, D. C. (1988). Single channel events and gating behavior of the cardiac gap junction channel. Proc. Natl. Acad. Sci. USA 85: 3431-3434.

DERMIETZEL, R., TRAUB, O., HWANG, T. K., BEYER, E., BEN- NETT, M. V. L., SPRAY, D. C., AND WILLECKE, K. (1989). Dif- ferential expression of three gap junction proteins in develop- ing and mature brain tissues. Proc. Natl. Acad. Sci. USA 86: 10148-10152.

EGHBALI, B., KESSLER, J., AND SPRAY, D. C. (1990). Expres- sion of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding con- nexin32. Proc. Natl. Acad. Sci. USA 87: 1328-1331.

FISHMAN, G. I., SPRAY, D. C., AND LEINWAND, L. A. (1990).

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

Molecular characterization and functional expression of the human cardiac gap junction channel. J. Cell Biol. 111: 589- 598.

FISHMAN, G. I., HER-ERG, E. L., SPRAY, D. C., AND LEIN- WAND, L. A. (1991). Expression of connexin43 in the develop- ing rat heart. Circ. Res. 68: 782-787.

FROHMAN, M. A., DUSH, M. K., AND IVLUTIN, G. R. (1988). Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 86: 899&9002.

GIMLICH, R. L., KUMAR, N. M., AND GILULA, N. B. (1990). Differential regulation of the levels of three gap junction mRNAs in Xerwpus embryos. J. Cell Biol. 110: 597-605. HERTZBERG, E. L., AND JOHNSON, R. G. (Eds.) (1988). “Gap Junctions,” A. R. Liss, New York.

KUMAR, M. M., AND GILULA, N. B. (1986). Cloning and characterization of human and rat cDNAs coding for a gap junction protein. J. Cell Biol. 103: 767-776. LEINWAND, L. A., SAEZ, L., MCNALLY, E., AND NADAL-GIN- ARD, B. (1983). Isolation and characterization of human myosin heavy chain genes. Proc. Natl. Acad. Sci. USA 80: 3716- 3720. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982). “Molecular Cloning,” Cold Spring Harbor Laboratory. Cold Spring Harbor, NY.

MILLER, T., DAHL, G., AND WERNER, R. (1988). Structure of a gap junction gene: Rat connexin32. Biosci. Rep. 8: 455-464.

NAYLOR, S. L., SAKAGUCHI, A. Y., SHOWS, T. B., LAW, M. L., GOEDDEL, D. V., AND GRAY, P. W. (1983). Human immune interferon gene is located on chromosome 12. J. Exp. Med. 67:1020-1027. PAUL, D. (1986). Molecular cloning of cDNA for rat liver gap junction protein. J. Cell Biol. 103: 123-134.

PEARSON, W. R., AND LIPPMAN, D. J. (1988). Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85: 2444-2448.

SHOWS, T. B. (1983). In “In Isozymes: Current Topics in Bio- logical and Medical Research” (M. C. Rattazzi, J. G. Scanda- lios, and G. S. Whitt, Eds.), Vol. 10, pp. 323-339, A. R. Liss, New York.

SHOWS, T. B., BROWN, J. A., HALEY, L. L., BYERS, M. G., EDDY, R. L., COOPER, E. S., AND GOGGIN, A. P. (1978). As- signment of the P-glucuronidase structural gene to the pter- q22 region of chromosome 7 in man. Cytogenet. Cell Genet. 21: 99-104.

SHOWS, T. B., SAKAGUCHI, A. Y., AND NAYLOR, S. L. (1982). In “Advances in Human Genetics” (H. Harris and K. Hirsch- horn, Eds.), Vol. 12, pp. 341-452, Plenum Press, New York/ London.

SHOWS, T., EDDY, R., HALEY, L., BYERS, M., HENRY, M., FUJITA, T., MATSUI, H., AND TANIGUCHI, T. (1984). Interleu- kin 2 (IL2) is assigned to human chromosome 4. Somat. Cell Mol. Gerzet. 10: 315-318.

WILLECKE, K., JUNGBLUTH, S., DAHL, E., HENNEMANN, H., HEYNKES, R., AND GRZESCHIK, K. (1990). Six genes of the human connexin gene family coding for gap junctional proteins are assigned to four different human chromosomes. Eur. J. Cell. Biol. 53: 275-280.

ZHANG, J.-T., AND NICHOLSON, B. (1990). Sequence and tissue distribution of a second protein of hepatic gap junction, Cx26, as deduced from its cDNA. J. Cell Biol. 109: 3391-3402.

the human connexin gene family of gap junction proteins: distinct chromosomal locations but similar...

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